Microsomal 5-ane-3β-hydroxysteroid oxidoreductase from pubertal rat leydig cells: Partial purification and characterization

Abstract

A 3β-hydroxysteroid oxidoreductase which acts on 5α(β)-reduced C 19 and C 21 steroids (5-ane-3β-hydroxysteroid oxidoreductase; 5-ane-3β-HSO) has been solubilized from pubertal rat Leydig cell microsomes and purified 300-fold by ion exchange and gel filtration chormatography. The partially purified enzyme is stable only in the presence of 0.4 M NaCl and appears to exist as a molecule having a molecular weight of 35,000 or as aggregates with a molecular weight in excess of 150,000. NAD + and NADH + are used exclusively as cofactors. The velocity of the steroid oxidation reaction was unaffected by either Ca 2+ or Mg 2+. The steroid oxidation reaction has a pH optimum between 8.0 and 8.5, a temperature optimum at 35°C and an activation energy of 12,850 cal/mol. The pH optimum of the steroid reduction reaction is 6.6. A variety of 5α-reduced C 19 and C 21 steroids can be utilized as substrates. Treatment of microsomes with phospholipase A; resulted in a 26 to 90% loss of enzyme activity, paralleling decreased microsomal phospholipid content, and suggesting a role for phospholipids in 5-ane-3β-HSO activity. Assays with combined substrates indicate that one enzyme is responsible for activities observed with 5α- and 5β-reduced C 19- and 5α-reduced C 21-3β-hydroxysteroids. Purification data indicate that the 5-ane-3β-HSO and the 5-ene-3β-hydroxysteroid oxidoreductase: isomerase are distinct enzymes.